The present invention relates to a biosensor facilitating rapid and highly accurate quantification of a measuring subject (a subject to be measured) contained in a sample in a simplified manner.
A biosensor has conventionally been proposed in the Japanese Laid-Open Patent Publication Hei 2-062952 as a system for simplified quantification of a specific component in a sample without diluting or stirring a sample solution.
This biosensor is produced by first forming an electrode system comprising a measuring electrode, a counter electrode and a reference electrode on an electrically insulating base plate by using a screen printing method or the like, and then forming an enzyme reaction layer comprising a hydrophilic polymer, an oxidoreductase and an electron mediator. If occasion demands, a buffer is added to this enzyme reaction layer.
When a sample solution containing a substrate is dropped on the enzyme reaction layer of the biosensor thus formed, dissolution of the enzyme reaction layer takes place, which in turn triggers a reaction between the enzyme and the substrate, causing reduction of the electron mediator. Upon completion of the enzyme reaction, the reduced electron mediator is oxidized electrochemically. The concentration of the substrate in the sample solution can be determined from the oxidation current value measured in this oxidizing step.
This biosensor can be used theoretically for measurements of various materials if an appropriate enzyme for each of the materials as the substrate is selected. For example, the use of glucose oxidase as the oxidoreductase can realize a biosensor for measurement of blood glucose level. This sensor is widely applied practically as a glucose sensor. The use of cholesterol oxidase as the oxidoreductase can realize a biosensor for measurement of serum cholesterol.
Serum cholesterol level which serves as the diagnostic standard is the sum of serum cholesterol and cholesterol ester concentrations. Since cholesterol ester cannot serve as a substrate for oxidation by cholesterol oxidase, an additional step of converting cholesterol ester into cholesterol becomes necessary in order to determine serum cholesterol concentrations as the diagnostic standard. Cholesterol esterase is known as an enzyme for catalyzing this process.
Inclusion of this cholesterol esterase together with cholesterol oxidase in the enzyme reaction layer can realize a biosensor for measurement of the total cholesterol concentration in serum.
In the biosensor having such constitution, the enzyme reaction layer is formed by dropping an aqueous reagent solution containing at least an oxidoreductase onto the electrode system and drying the dropped solution. Such biosensor has a problem, when the amount of reagents is large, that the reaction layer is not dissolved quickly after dropping of the sample solution thereonto so that the measurement takes a long time.
Particularly in the cholesterol sensor, two kinds of enzymes, cholesterol oxidase and cholesterol esterase, must be contained in the enzyme reaction layer, as described previously. Therefore, the enzyme reaction layer contains a considerably large amount of reagents, so that dissolution of this layer takes a significantly long time after dropping of the sample solution, which makes it impossible to make rapid measurement.
In order to solve the above-mentioned problem, a biosensor has been proposed in Japanese Laid-Open Patent Publication 2000-039416. The biosensor comprises an insulating base plate, an electrode system including at least a measuring electrode and a counter electrode formed on the base plate, a cover member which is integrated into the base plate and forms a sample solution supply pathway for supplying a sample solution to the electrode system between the cover member and the base plate, and a carrier comprising a fiber for supporting reagents containing at least an oxidoreductase, wherein the carrier is disposed in the sample solution supply pathway.
Also, this biosensor according to another embodiment comprises an insulating base plate, an electrode system including at least a measuring electrode and a counter electrode formed on the base plate, and a carrier comprising a fiber for supporting reagents containing at least an oxidoreductase, wherein the carrier is fixed with an adhesive in the vicinity of the electrode system.
In this biosensor, however, the height of the sample solution supply pathway is required to be at least equal to or greater than the thickness of the carrier, since the carrier is disposed in the sample solution supply pathway. Moreover, the biosensor actually has a structure that the carrier and the electrode system are disposed to face each other, thereby requiring a certain space between the electrode system and the surface of the carrier in order to prevent the contact between them. Thus, the height of the sample solution supply pathway is required to be further greater.
For these reasons, when the carrier is disposed in the sample solution supply pathway, the biosensor requires a sample solution in a relatively large amount in order to fill the sample solution supply pathway with the sample solution. The amount is determined by multiplying the carrier volume calculated from the external dimensions thereof by the porosity of the carrier to obtain a product and by adding to the product the above-mentioned space between the electrode system and the surface of the carrier.
Meanwhile, it is more preferable that the porosity of the carrier is higher in order to improve dissolution of the reagents.
From such characteristics as described, the above-described biosensor tends to need a larger amount of the sample solution for the measurement than the biosensor without a carrier.
Also, especially when the sample solution is blood and the biosensor is provided with a function of filtering blood corpuscle components with a porous material such as a filter paper, the porous material for filtering the blood corpuscle components has sometimes made it difficult for the filtered sample solution to invade into the sample solution supply pathway, since the carrier is disposed in the sample solution supply pathway.
In view of the above problems, an object of the present invention is to provide a biosensor in which dissolution of reagents is improved to enable prompt quantification and reduction of the amount of a sample solution. Further, another object of the present invention is to provide a biosensor in which the sample solution can be introduced readily.
A biosensor in accordance with the present invention comprises an electrically insulating base plate; an electrode system including at least a measuring electrode and a counter electrode formed on the base plate; a cover member, which is integrated into the base plate and forms a sample solution supply pathway for supplying a sample solution to the electrode system between the cover member and the base plate; a reaction reagent system including at least an electron mediator and an oxidoreductase; and a carrier supporting at least the electron mediator or the oxidoreductase of the reaction reagent system, wherein the carrier is partially or wholly disposed outside the sample solution supply pathway.
In the biosensor in accordance with the present invention, it is effective that the carrier has a region supporting at least the electron mediator or the oxidoreductase disposed closer to the electrode system than a region supporting none of the reaction reagent system.
Also, it is effective that the oxidoreductase is cholesterol oxidase.
Further, it is preferable that the reaction reagent system contains an enzyme capable of hydrolyzing cholesterol ester.
It is effective that the enzyme capable of hydrolyzing cholesterol ester is cholesterol esterase.
Furthermore, it is preferable that the reaction reagent system contains a surfactant.
It is effective that the carrier is porous and, further, that the carrier is fibrous or comprises a fiber.
While the novel feature of the invention are set forth particularly in the appended claims, the invention, both as to organization and content, will be better understood and appreciated, along with other objects and features thereof, from the following detailed description taken in conjunction with the drawings.